One of the major scientific challenges in vaccinology today is the development of an effective AIDS vaccine Evidence has been accumulated that MHC class I restricted CD8+ cytotoxic T lymphocyte (CTL) responses may provide protection against HIV infection. As Phase I/II clinical trials for testing of candidate AIDS vaccines progresses, canarypox-based vaccine strategies have elicited anti-HIV-1 CTL in a significant number of uninfected immunized volunteers. Although we have begun to better understand the specificity and duration of the elicited CTL reactivities, little is still known about the qualitative aspects of these responses. Based on preliminary studies performed in association with recently completed trials, the specific aims of this proposal are designed to test the major hypothesis that vaccine induced anti-HIV CTL responses are qualitatively different from the responses elicited by natural HIV infection. Studies are designed to characterize CTL lines, and ultimately CTL clones, from antigen-specific in vitro stimulation (IVS) cultures with respect to avidity, TcR receptor breadth, as well as cytolytic and non-cytolytic virus suppressive reactivities against autologous lymphocyte target cells infected with primary isolates representing genetically diverse viral clades. Avidity will be measured both by conventional peptide titration as well as peptide/MHC tetramer binding. Tetramers will also be utilized to track epitope-specific CTL and their maturation stage in PBMC from vaccinees. The capacity of canarypox vectors to bias the CTL responses of vaccinees will also be studied at the level of in vitro CTLp amplification, using a panel of different HIV-expressing recombinant vectors in different APC-based antigen-specific stimulation strategies as well as HIV-1-specific activation. Lastly, comparative studies will be performed to determine whether the quality of vaccine- induced CTL reactivities more closely resemble CTL responses that develop during the acute phase of HIV-1 infection, especially in association with structured early treatment interruption before the immune depletion associated with prolonged viral stimulation and infection become manifest. By yielding new insights into the quality of vaccine-induced CTL, these studies could greatly impact the further development of vector-based vaccines.